1. Field of the Invention
The present invention is a method and apparatus that provides an inexpensive, quick, and portable means to separate motile sperm from a sperm sample. The invention passively filters the motile sperm using a nucleopore membrane.
2. Background Art
Artificial insemination has become a much more frequent procedure due to a variety of sociological, economic, and perhaps environmental reasons. The number of inseminations in the United States and worldwide has been increasing, and will likely continue to increase. A number of causes have been cited for this increase. The age of childbearing has increased, as people are delaying children and marriage. The increased age lowers the probability of fertilization for both men and women. More and more women are also raising children by themselves without a partner, and electing artificial insemination as a means of fertilization. In addition, those that would otherwise not be able to have children now have a medical option that is relatively affordable. An additional factor may be that the sperm count among males has been declining, making fertilization more difficult. And finally, environmental factors have been blamed for decreased fertility of both men and woman.
There are currently a wide array of artificial insemination methods such as, intracervical, intrauterine (IUI), intratubular and direct intraperitoneal (DIPI) insemination, gamete intrafallopian transfer (GIFT), in vitro fertilization and embryo transfer (IVFET), zygote intrafallopian transfer (such as ZIFT, PROST and TET), peritoneal oocyte and sperm transfer (POST), and sex selection, among others. As technology advances, other methods are certain to follow, however, regardless of the process, high motile sperm are preferred. And, most of the facilities that perform the insemination do not have the resources to separate motile sperm, requiring a separate visit to a facility that possess the separation means.
As an example, the Intrauterine Insemination (IUI) and In Vitro fertilization (IVF) methods attempt to recreate the reproductive process by placing sperm and eggs together in an environment conducive to fertilization, either in the womb or outside the womb. The fertilization process requires the sperm to actively invade the egg and commence fertilization. Motile sperm are much more likely to penetrate the egg.
A typical semen sample contains materials such as paternal plasma, protein, leukocytes, spermdecapitation factors and other extraneous materials, and dead, agglutinated or nonviable spermatozoa. These materials are known to interfere with successful fertilization, and with the successful maintenance of a fertilized ovum in the female patient. As an example, seminal plasma can cause severe uterine cramping, and in a worst case results in spontaneous abortion of the fertilized ovum. Thus, it is desirable to remove those spermatozoa having relatively low motility or possessing unhealthy, damaged or abnormal membranes.
The total number of sperm in an ejaculate is a measure of fertility, however, the percentage of motile sperm is more important, especially when considering alternate reproductive means.
Sperm are categorized according to the their exhibited motility, as exhibited by Table 1.
The percentage of motile sperm showing progressive swimming movements is a measure of the fertility of the sperm sample. The higher the percentage, the higher quality of the sperm sample, and the greater the likelihood that the sample will achieve fertilization.
A high quality sperm sample is important for many reasons. The process of artificial insemination is not only costly economically, but is psychologically expensive. Unsuccessful attempts have devastating effects on the patients.
Higher quality sperm samples are also important considerations when the sample is subject to freezing or aqueous dilution, because these processes tend to kill or weaken the sample. Thus, only the highest quality sperm may survive the processing procedures to which the sperm are subjected.
In summary, higher viability spermatozoa are more likely to lead to successful fertilization and impregnation. The higher quality sperm are also more likely to survive freezing (cryopreservation) and other processing procedures.
In order to reduce the aforementioned problems, attempts have been made to provide a simple, portable, and inexpensive method and device for the filtration of sperm. However, the prior art devices and methods had significant disadvantages and shortcomings.
Various methods of selecting the more active sperm have been utilized in the past, such as the swim up, swim down and Percoll density gradient centrifugation techniques. Swim-up methods are commonly used to process fresh or frozen specimens for the IUI and IVF procedures. The sperm is placed in a medium and subjected to a centrifuge process. The more motile sperm swim to a level where they can be extracted. Such methods employ multiple tube and centrifugation steps that are time consuming and can lead to a low recovery of motile sperm.
One prior art device used for separating motile sperm from a sperm sample is the migration-sedimentation chamber. This system uses a culture medium with the sperm sample suspended in a medium, and requires a centrifuge to rapidly spin the chamber to separate motile sperm. And, because it is an active process, it increases the likelihood that motile sperm will be excluded from the sample set or damaged during the process, producing a lower recovery sample.
In operation, the sperm sample is placed in a migration-sedimentation chamber, preferably in the lower well portion. The chamber is subjected to centrifugation, and the re-suspended sperm pellet is transferred to the gallery segment of the chamber. The portion of sperm in the well is the motile portion, and is used for insemination.
U.S. Pat. No. 5,185,246 (""246) is a method for semen analysis employing a membrane separation. This patent discloses a method to separate the particulate (cells, spermatozoa, and other particulate) in the whole semen from the seminal plasma which contains many soluble compounds, proteins, hormones, small molecules and electrolytes. The pore size is specifically designed for such a separation, and all sperm cells are blocked from passing through the membrane. The driving force of the ""246 patent is a vacuum, and the sperm cells are stuck to the filter, dead and immobile, amenable only for in-situ testing or further disruption. A specific volume, preferably 100 microliters with various aqueous dilutions is employed. The separation apparatus of the ""246 patent is a well filter plate that is used for multiple testing in the same round of processing.
The ""246 patent is related to tests of andrological significance that need be performed on either the seminal plasma (in the absence of sperm cells) or on the surface of the sperm cells (in the absence of the seminal plasma). But, the cells in these cases need not be viable, need not be motile, and need not be freely dispersed in an aqueous buffered medium for retrieval. The ""246 patent discloses a device for providing easier diagnostic access to specific components of the semen sample.
Another such invention is described in U.S. Pat. No. 5,575,914, that discloses a conduit with glass wool as a strainer mechanism. The glass wool is compressed to a density sufficient to permit passage of more motile sperm while blocking lesser or non-motile sperm. In the preferred embodiment, the conduit is a nested pair of tubes that keeps the glass wool density consistent.
The invention of U.S. Pat. No. 4,009,260 describes the fractionation of sperm through layered migration of various layers with differing densities. The more motile sperm penetrate deeper into the layers. Similarly, U.S. Pat. No. 4,007,087 also discloses the fractionation via layered solutions contacting the sperm sample.
U.S. Pat. No. 5,427,946 discloses a channeling apparatus, where there are inlet ports, flow channels, and nesting chambers. The sperm sample is applied at the inlet port, and only the motile sperm are capable of reaching the chambers.
In summary, problems heretofore exhibited in the art include having to purchase and maintain additional equipment such as a centrifuge or vacuum. Prior methods required taking trips to multiple sites to perform the separation or centrifuge process. Other methods required extensive time to separate the motile sperm. The prior devices also required on-site sampling and proper handling by skilled individuals. In addition, prior art methods that used active separation schemes removed motile sperm and reduced the recovery lot.
What is needed is a method and apparatus that can passively isolate higher viability spermatozoa from the spermatozoa having relatively lower motility. The device should be inexpensive, portable, and easy-to-use. The method screens out the lesser sperm and other compounds leaving only high quality sperm for insemination. What is needed is an efficient device and method of removing the less motile sperm without costly or time-consuming procedures, and this new system should provide a quick and inexpensive way to increase the likelihood of fertilization. Furthermore, the sperm separation should be available on-site where the insemination is to take place to avoid undue delay and travel with separate facilities that perform such functions.
The present invention has been made in consideration of the aforementioned background. It is therefore an object of the present invention to provide a system for conveniently, quickly, and inexpensively separating motile sperm from a sperm sample. This invention most generally relates to a method and an apparatus of straining sperm. In particular, it relates to a method of straining and separating motile sperm from a larger sample set without using additional machinery or devices.
The driving impetus of the present invention is the ability of quality sperm to move and swim through the membrane. The non-motile sperm and leukocytes move slightly, if at all, and therefore do not penetrate the membrane. No other force, such as a vacuum, suction, or gravity is required. Any artificially induced stress or pressure would defeat the purpose of the present invention by forcing non-motile sperm through the membrane and also possibly damaging the motile sperm by the applied force. The natural, self-actuated migration of the more motile sperm in the semen bath assures that the sperm that do encounter and work their way through the membrane will have a significantly higher average motility index than the remaining sperm.
The present invention further provides a method and apparatus for the separation of motile swimming cells from the dead/non-motile spermatozoa and from white blood cells that contaminate semen in case of inflammatory events. The present invention ascribes no significance to the separation of seminal plasma from the particulate matter since it is not a desired component that needs special care and attention.
The method and apparatus of the present invention is for the separation of the xe2x80x98betterxe2x80x99 sperm, unattached to membranes or anything else, and in a form immediately available for retrieval and subsequent use in artificial insemination by means of a filtering membrane.
The motile sperm that penetrate the membrane may be supported in a buffered medium. After an incubation period, the motile sperm is immediately ready for functional analysis and the various methods of artificial insemination.
The present device and methodology offers a rapid and efficient method for improving the sperm quality as a routine sperm preparation for artificial insemination. This device is a one-step system that enables the recovery of high-quality motile sperm in minimal time. No washing or centrifugation required, and the sperm preparation can be done in the office or at the desired location.
One preferred embodiment of the present invention includes a cell strainer unit with a 5 micron nylon mesh size and with an openable lid. This strainer is compatible with a 1 ml dish and has a screw cap. The 1 ml dish is used to hold the medium and retain the cell strainer unit when properly engaged. A disposable plastic pipette is used to place the sperm sample into the cell strainer, and a 1 ml disposable syringe with needle is used to place the medium into the dish and to extract the sperm sample when incubation is completed.
The method of operation of this first preferred embodiment comprises placing 1 ml of a medium the container or dish using the syringe with the needle. The user then puts 1-1.5 ml of liquefied semen into the strainer or membrane unit using a disposable plastic pipette. The lid on the strainer is closed, capturing the membrane or mesh securely in place, and the strainer unit is secured onto the dish. The unit is left for 30-45 minutes for incubation at 37xc2x0 C. in 5% CO2. At the end of the incubation period, the user can remove the strainer unit from the dish and discard the cell strainer. The sperm sample remaining in the dish is the motile sperm sample and the user can carefully aspirate all media from the dish, using the syringe with needle. The recovered sperm in media is ready to use for artificial insemination. For IUI applications, it is necessary to remove the needle from the syringe and attach the catheter, and perform the IUI as normally done in the industry.
Tables 3, 4 and 5 below illustrate three actual examples of the original semen sample and the results of the Swim-Up Method of the prior art, as compared to the Cell-Strainer Method.
It will be readily apparent that all three methods result in reductions in total sperm count, however the total is still very high, viewed statistically, relative to the likelihood of achieving fertilization. More importantly, in all cases the motility percentage is increased significantly. As is illustrated, in five out of six cases, the invention provided greater improvement in motility than the Swim-up method of the prior art.
The present invention is for a simple, inexpensive and disposable system for extracting motile sperm and preventing inferior sperm from entering the reproduction process. Artificial reproduction methods are costly and time-consuming procedures, and carry highly emotional overtones upon failure. It is important to increase the likelihood of successful reproduction, and the filtering of sperm is common in the industry. The prior methods of filtering out the motile sperm involved centrifuges and swim-up methods to extract the motile sperm. These methods are time-consuming and may lead to a low recovery of motile sperm, requiring additional equipment and steps in the fertilization process. The additional equipment and time lead to a higher overall cost.
One object of the present invention is to separate motile sperm from the non-motile sperm or leukocytes. Another object of the invention is to provide an inexpensive and easy to use device, such that each patient can use a clean sperm filter.
The membrane of the present invention is large enough to allow for the passage of sperm through the membrane. In the preferred embodiment, a 5-8 micron pore size is utilized.
Yet an additional object of the invention is to provide a membrane apparatus that completely encloses the sample sperm before placing the enclosed sample into the container medium. This xe2x80x98tea bagxe2x80x99 approach provides a maximum surface area for the motile sperm to disperse from the sample into the medium.
A further object is to provide a sperm strainer system that can be used for animals as well as humans. In the preferred embodiment, human sperm is considered, however the invention would work equally well with other sperm without deviating from the scope of the invention. With other types of sperm, different sizes of mesh, and different mediums would be appropriate.
It is an object of the invention to provide a complete and portable sperm separation system. The invention can be packaged as a kit, and allow a fast and easy separation of the motile sperm. The kit would be not only cost effective, but can be used on locations that lack access to additional laboratory equipment required by the prior methods.
An additional object of the invention is to provide a sperm strainer apparatus for separating motile sperm from a sperm sample, where the sperm strainer apparatus comprises a container with a disposed medium, a membrane assembly, wherein the membrane assembly has a nucleopore membrane for passively separating the motile sperm from the sperm sample, and wherein the membrane assembly engages the disposed medium.
Yet a further object of the invention is to provide a sperm strainer apparatus having a lid attachable to the container.
An additional object of the invention is to provide a sperm strainer apparatus where the container has a threaded portion for engaging a mating threaded portion of the membrane assembly.
An object of the invention further includes a sperm strainer apparatus where the nucleopore membrane is 5-8 microns.
An additional object of the invention provides a sperm strainer apparatus wherein the nucleopore membrane is vertically disposed within the container.
A further object is for a sperm fractionation apparatus having a membrane assembly that fits within the container and supports the nucleopore membrane at an angle relative to the bottom surface of the container and extending downward substantially to the bottom surface of the container.
An object of the invention includes where a sperm fractionation apparatus has a membrane assembly that fits within the container and rests on a plurality of stand-offs that create a gap between a bottom surface of the container and a bottom surface of the membrane assembly.
A further object is a method of separating motile sperm from a sperm sample comprising the steps of contacting the sperm sample to a first surface of a nucleopore membrane having pores of 5-8 microns in diameter through which motile sperm can generally migrate freely, contacting a receiving medium suitable for sustaining live sperm to the opposing surface of the nucleopore membrane, incubating the sperm sample and receiving medium in contact with the membrane at ambient pressure and absent application of centrifugal effects until a sufficient population of sperm have migrated to the receiving medium.
An additional object is a device for separating motile sperm comprising a container and a membrane assembly, with a nucleopore membrane being supported by the membrane assembly. The container is divided into two chambers by the membrane assembly wherein the receiving medium can be contained in a first chamber in contact with the first surface of the nucleopore membrane and the sperm sample is contained in a second chamber in contact with the second surface of the nucleopore membrane.
A further object is a method of separating motile sperm, wherein the incubating is conducted for approximately 30 minutes. And, an object includes a method of separating motile sperm, wherein the incubating is conducted at a temperature of approximately 37xc2x0 C.
And yet another object is for a kit for the fractionation of motile sperm from a sperm sample by incubation at ambient pressure without the application of centrifugal effects. This kit comprises, a container; a receiving medium; a membrane assembly configured with a nucleopore membrane having pores of 5-8 microns diameter, the container being divisible into two chambers by insertion of the membrane assembly wherein the receiving medium can be contained in a first chamber in contact with the first surface of the nucleopore membrane and the sperm sample can be contained in a second chamber in contact with the second surface of the nucleopore membrane, means for inserting and removing the receiving medium, and means for inserting and removing the sperm sample.
Object where a kit for the fractionation of motile sperm, further comprising a lid for covering the container.
An additional object is for a kit for the fractionation of motile sperm, wherein the means for inserting and removing the receiving medium is a syringe for displacing the medium.
And yet a further object includes kits for the fractionation of motile sperm the means for inserting and removing said sperm sample comprising a pipette.
A second embodiment of the present invention is for a user-friendly device that does not require a screw cap. Eliminating the screw cap assembly and the mounting and removal of the membrane results in a simpler and more expedient gathering process.
While an improvement to the prior art, the first embodiment of the invention comprised a larger container with a sperm compartment of approximately 1 ml, and a cap that attached a membrane to the rim of the container. The first embodiment had numerous steps, namely mounting the membrane, screwing the cap, allowing for the separation while maintaining the container in a stable position, removing the cap, discarding the membrane, and then extracting the sample.
As shown in Table 6, a fill matrix of relationships for various dimensions of the compartment is shown relative to volume. The matrix is demonstrated for a single compartment, and generally the other compartment would be asymmetric. Although the preferred compartment size is 2 ml compartments, other dimensions may be utilized as shown. The parameters include the volume (ml), the diameter (cm), the short length (cm) and the long length (cm). Based on sample trials, the preferred size was a 2 ml volume with a dimension of 2 cm, a short length of 0.537 and a long length of 0.737.
The second embodiment seeks to optimize the sperm flow within the compartments and increase the collision rate between the sperm cells and the membrane. One of the principles of the second embodiment is that the depth (thickness of each compartment) is always significantly smaller than the diameter, so that sperm cells from the farthest points in the semen compartment would not require a long linear movement towards the membrane. This enhances the collision rate between cells and the membrane, and shortens the equilibration time between the two compartments. At the same time the depth is large enough so that the tip of a needle or any other device used to load or extract the sample does not rupture the membrane.
Another object of this embodiment is the need to do much of the steps in reverse order when extracting the improved sperm preparation from the sperm compartment. It is also an object to eliminate several of the steps of the sperm separation process.
A further object is the reduction in container volume and suspension medium, so no concentration step (centrifugation) is necessary before a suspension is available for insemination
The second embodiment also describes the membrane manufactured of polycarbonate with 4-8 um diameter holes. Pore diameters lower than 4-6 um, cells would not pass at all, but at higher pore diameters lymphocytes could passively pass through the larger diameter holes. The membrane is produced from a non sperm-toxic polymer, such as polycarbonate.
An additional object of the second embodiment is the orientation of the components. The preferred orientation is to have the membrane in the vertical position during the separation incubation. This is preferable because in the horizontal position non-motile cells and round cells may move passively through the pores.
A further object of the second embodiment is to produce a resultant high concentration suspension at the end of the procedure. Using a larger sperm sample and a lesser medium within the confines of the second embodiment eliminates the need for a concentration step (eg: centrifugation) before a suspension is available for insemination.
However, in order to utilize the vertical oriented membrane the device cannot be loaded in that position because the liquid mass would equilibrate between the two compartments before the loading is completed. Thus, the device is designed to lay with the membrane horizontal while loading, and then turned to the vertical orientation for the duration of the incubation.
An object of the second embodiment is to provide quick access to the compartments of sperm separation device such that the initial sperm sample can be injected into the device on an entry port and a more motile sperm sample can be extracted from an output port. The injection and extraction do not require any additional steps. Such ports may be angled needle guides.
A further object is to provide a closure means for sealing the input and output ports to avoid spillage and allow for transport. And an additional object includes the product design in such as manner as to be freestanding while the separation process occurs.
A final object is to generate a quality sperm density that is ready to use, unless derived from oligzoospermic patients.
The present invention describes a sperm fractionation apparatus for separating motile sperm from a sperm sample comprising a container having a sperm sample compartment and a motile sperm compartment with an access means into both of the compartments. There is a non sperm-toxic polymer membrane separating the sperm sample and motile sperm compartments through which the sperm sample can generally migrate. And, there is a receiving medium suitable for sustaining the motile sperm in the motile sperm compartment.
The sperm fractionation apparatus has a non sperm-toxic membrane, which may be a polycarbonate, and the pores of the non sperm-toxic membrane are 4-8 microns in diameter. The sperm fractionation apparatus further comprises a needle stop plastic strip adjoining both sides of the polycarbonate membrane. There is also a plug assembly for closing said access means.
A preferred embodiment has a sperm fractionation apparatus wherein the non sperm-toxic membrane is vertically disposed within the container during incubation. The sperm fractionation container in one embodiment is a cylinder with the access means on both ends of the cylinder, and both ends of said cylinder are outwardly projecting. The diameter of the cylinder is significantly larger than a length of the sperm sample and motile sperm compartments. Finally, the container has outwardly projecting flanges extending beyond both ends of the cylinder so that it can be free-standing.
The present invention is a sperm fractionation apparatus for separating motile sperm from a sperm sample comprising an integral container having a sperm sample compartment and a motile sperm compartment with an access means into both of the compartments, wherein a height of the sperm sample compartment and the motile sperm compartment exceeds a width of the sperm sample compartment and the motile sperm compartment. There is a non sperm-toxic polymer membrane separating the semen and motile sperm compartments through which the sperm sample can generally migrate. And, there is a receiving medium suitable for sustaining the motile sperm in the motile sperm compartment.
In one embodiment the sperm fractionation apparatus the access means is an angled orifice. This prevents accidental damage to the membrane. Also, the container has projecting flanges in order to provide a stable base for the container when placed in a horizontal position. The container also has projecting flanges in order to provide a stable base for the container when placed in a vertical position. The device also uses a Teflon plug assembly for closing the access means. In a preferred embodiment each of the sperm sample compartment and the motile sperm compartment has a volume of 2 ml.
One method of separating motile sperm from a sperm sample using a sperm separation device, comprises the steps of plugging both access ports of the sperm separation device, placing the sperm separation device in a horizontal position with a motile sperm compartment facing upwards, opening the motile sperm compartment and filling with a buffer medium, closing said motile sperm compartment, rotating the device so a sperm sample compartment is facing upwards, opening the sperm sample compartment and introducing a sperm sample, closing the sperm sample compartment and rotating the device so a membrane in the device is vertically orientated, allowing the sperm sample to incubate, and rotating the device so the motile sperm compartment is facing upwards and extracting the motile sperm. The time in which incubating is conducted is for approximately 45 minutes.
In summary, the present invention in its various embodiments provides a simple and cost-effective apparatus and method for collecting, separating and transporting motile sperm. The apparatus requires little or no assembly, it fast and easy to operate. It optimizes collision between sperm cells and the membrane and produces better sperm density for extraction. There is no need for an external vacuum or centrifuge, as the sperm is separated passively.
Still other objects and advantages of the present invention will become readily apparent to those skilled in this art from the following detailed description, wherein a preferred embodiment of the invention is shown and described, simply by way of illustration of the best mode contemplated for carrying out the invention. Other objects, features and advantages are apparent from the descriptions presented in conjunction with the accompanying drawings.